EP3525807B1

EP3525807B1 - Inhibitor of rna polymerase ii

Info

Publication number: EP3525807B1
Application number: EP17797991.1A
Authority: EP
Inventors: Catharina Svanborg; Nina FILENKO; Ines AMBITE
Original assignee: Selectimmune Pharma AB
Current assignee: Selectimmune Pharma AB
Priority date: 2016-10-17
Filing date: 2017-10-16
Publication date: 2021-12-01
Anticipated expiration: 2037-10-16
Also published as: US20190240287A1; US20210069291A1; SG11201903336UA; GB201617548D0; US11752192B2; AU2017344453B2; AU2017344453A1; WO2018073725A1; DK3525807T3; EP3525807A1

Description

The present invention relates to factors and moieties that can modulate host protein expression, for example, a factor having immunosuppressant activity, and to their use in therapy.

Background to the Invention

The multi-subunit RNA polymerase II complex (Pol II) is essential for protein expression in eukaryotes. Transcription cycle has been extensively characterized to show that around 10% of all expressed genes are involved in transcriptional regulation, assembly and control of the Pol II complex. In eukaryotic cells, RNA polymerase II catalyzes the synthesis of mRNA and small nuclear RNA. Pol II transcription cycle consists of several stages, termed preinitiation, initiation, promoter clearance, during which Pol II pauses at the promoter proximal site, followed by escape from pausing, productive elongation and termination.
For specificity, the assembly of Pol II is tightly controlled and the efficiency of transcription is modified at individual promoters by activators and repressors, as well as general or specific transcription factors. The carboxy-terminal domain (CTD) of the largest Pol II subunit, Rpb1, contains a heptamer sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 repeated 52 times in mammalian cells. During the transcription cycle, CTD is subject to continuous structural remodeling by kinases and phosphatases and serves as a platform for binding and release of numerous regulatory proteins. The recruitment of Ser2-specific kinase activity in the form of positive transcription elongation factor b (P-TEFb) is regarded to be a critical step in the activation of promoter-proximally paused Pol II, facilitating its release from pause sites. P-TEFb is composed of cyclin-dependent kinase 9 (CDK9) and its regulatory cyclin T1. The more recently discovered Cdk12 phosphorylates CTD of RNA polymerase in the middle and 3' end of genes. P-TEFb is a general transcription factor required for efficient expression of most cellular genes, therefore, its activity is accurately mediated with positive regulator bromodomain protein Brd4, and negative regulators noncoding 7SK snRNA and the HEXIM1 protein. It was shown that P-TEFb recruits PAF1 to Pol II complex, which is followed by CDK12 recruitment by PAF1 (Yu et al., Science 2015. 350(6266:1383-6). De-phosphorylation of Ser2-phosphates is done by phosphatases FCP1 (TFIIF-dependent CTD phosphatase 1) and Cdc14.
The host Pol II transcription machinery is targeted by bacteria, as first described for Escherichia coli strains that establish an asymptomatic carrier state in the human urinary bladder (Lutay et al., J. Clin Invest. 2013, 123(6) 2366-79). Asymptomatic bacteriuria actively modifies the host response by inhibiting Pol II dependent transcription, including pathology-generating signaling pathways in the host (Lutay et al., 2013 supra.). In 24 hours after human inoculation with the prototype ABU E. coli strain 83972, more than 60% of all genes were suppressed; this inhibition was verified by infection of human cells. Among different ABU strains (n=75), 37% were strongly inhibitory compared to 17% of APN strains (n=88). The symbiotic relationship between ABU strains and their hosts is also influenced by a lack of virulence factors, resulting from virulence gene attenuation in these strains.
These findings indicate that suppression or inhibition of RNA polymerase II may be an effective method for modulating the immune system, in particular by acting as modulators of gene expression. There is frequently a need to modulate gene expression, in particular of the immune system either by stimulation or suppression in connection with a wide variety of therapeutic applications.
Immunosuppressants are required in a wide a variety of therapies. This includes for instance, the prevention of rejection of transplanted organs or tissues such as bone marrow, heart, kidney or liver, the treatment of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis, lupus, sarcoidosis, Crohn's disease, pemphigus and ulcerative colitis.
However, as a broad spectrum suppressor of protein expression, Pol-II inhibitors may find application in any therapy where host protein is over-expressed or problematic. Thus Pol-II inhibitors may also be used in the treatment of inflammatory disease such as asthma, or in the treatment of auto-inflammatory disease such as Behcet's disease, FMF, NOMID, TRAPS or DIRA. It has also been suggested that host-directed immunomodulatory therapies can be used in the treatment of infections, whereby natural mechanisms in the host are exploited to enhance therapeutic benefit. In this case, the objective is to initiate or enhance protective antimicrobial immunity while limiting inflammation-induced tissue injury. Such a mechanism may help to address the increase or antimicrobial resistance as bacteria become resistant to conventional antimicrobial drugs over time.
Earlier attempts to characterize the mechanism of Pol II inhibition by the ABU by comparison of whole genome sequences of E. coli 83972 and the uropathogenic strain, E.coli CFT073 have failed to identify specific factors associated with this (I. Amibite et al. Pathogens, 2016, 5, 49; doi: 10.3390), indicating that this is not a simple matter.
However, as a result of the serendipitous occurrence of attenuated Pol II inhibitory activity in a re-isolate, obtained from a patient inoculated with a prototype ABU, the applicants have been able to make a significant breakthrough in identification of the factors which may be useful in immunosuppression.

Summary of the Invention

According to a first aspect of the invention, there is provided an inhibitor of host RNA polymerase II (pol II inhibitor) for use in therapy, wherein said inhibitor is a moiety which targets a protein selected from PAF1C or CDK12, and wherein said inhibitor is an E. coli NlpD protein, or a an active variant thereof, the variant comprising an amino acid sequence having at least 70% sequence identity with the native protein, or an E. coli Sigma S protein or an active variant thereof, the variant comprising an amino acid sequence having at least 70% sequence identity with the native protein, or an active fragment of either of these, the active fragment comprising SEQ ID NO 3.
Moieties which target these proteins may be known in the art or may be identified or designed using conventional screening methods.
Specific examples of such moieties may be obtained from bacteria, such as the commensal bacteria or asymptomatic carriers such as asymptomatic bacteriuria.
As a result of the isolation of a spontaneous, loss of function mutant of the ABU strain E. coli 83972, the applicants have identified bacterial proteins able to act as direct inhibitors Pol II phosphorylation in infected hosts. Mutations were localized by comparative genome sequencing of the E. coli 83972 WT and mutant strain and identified sequence variants were systematically introduced into the E. coli 83972 WT strain for analyses of effects on Pol II phosphorylation.
The TATA box binding protein Sigma S was shown to bind human TATA box DNA and to competitively inhibit the binding of human TBP, thus incapacitating the Pol II pre-initiation complex. Furthermore, NlpD, which regulates Sigma S expression, stimulated the degradation of PAF1C, which recruits the kinase CDK12 to the Pol II phosphorylation complex. Both proteins entered host cells. The results identify a novel mechanism used by bacteria to regulate fundamental host cell functions, such as the transcriptional activity at sites of infection.
In a particular embodiment, the inhibitor is a bacterial Sigma S protein or an active variant thereof, the variant comprising an amino acid sequence having at least 70% sequence identity with the native protein, or an active fragment of either of these, the active fragment comprising SEQ ID NO:3. An example of such a protein is SEQ ID NO 2 or a truncated form of SEQ ID NO 2.
As used herein, the term 'fragment' refers to any portion of the given amino acid sequence which will shows Pol II inhibitory activity. Fragments may comprise more than one portion from within the full-length protein, joined together. Portions will suitably comprise at least 5 and preferably at least 10 consecutive amino acids from the basic sequence.
Suitable fragments will include deletion mutants comprising at least 10 amino acids, for instance at least 20, more suitably at least 50 amino acids in length or analogous synthetic peptides with similar structures. They include small regions from the protein or combinations of these.
The fragment will be a peptide which inhibits binding of TBP to Sigma S. The fragment comprises amino acids 149-183 of SEQ ID NO 2, shown in bold type in the above sequence, which forms SEQ ID NO 3.
Certain such pol II inhibitors will be novel and these form an aspect of the invention.
They may be used in a range of therapeutic applications as discussed above, to suppress protein expression. In particular, they may be used as immunosuppressant, anti-inflammatory or anti-infection (such as antibacterial) agents.
The expression "variant" refers to proteins or polypeptides having a similar biological function but in which the amino acid sequence differs from the base sequence from which it is derived in that one or more amino acids within the sequence are substituted for other amino acids. Amino acid substitutions may be regarded as "conservative" where an amino acid is replaced with a different amino acid with broadly similar properties. Non-conservative substitutions are where amino acids are replaced with amino acids of a different type.
By "conservative substitution" is meant the substitution of an amino acid by another amino acid of the same class, in which the classes are defined as follows:

Class Amino acid examples

Nonpolar: A, V, L, I, P, M, F, W

Uncharged polar: G, S, T, C, Y, N, Q

Acidic: D, E

Basic: K, R, H.
As is well known to those skilled in the art, altering the primary structure of a polypeptide by a conservative substitution may not significantly alter the activity of that polypeptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the peptide's conformation.
Non-conservative substitutions are possible provided that these do not interrupt the function of the DNA binding domain polypeptides. Broadly speaking, fewer non-conservative substitutions will be possible without altering the biological activity of the polypeptides. Determination of the effect of any substitution (and, indeed, of any amino acid deletion or insertion) is wholly within the routine capabilities of the skilled person, who can readily determine whether a variant polypeptide retains the fundamental properties and activity of the basic polypeptide. For example, when determining whether a variant of the polypeptide falls within the scope of the invention, the skilled person will determine whether complexes comprising the variant retain biological activity (e.g tumour cell death) of complexes formed with unfolded forms of the native protein and the polypeptide has at least 60%, preferably at least 70%, more preferably at least 80%, yet more preferably 90%, 95%, 96%, 97%, 98%, 99% or 100% of the native protein.
Variants of the polypeptide comprise or consist essentially of an amino acid sequence with at least 70% identity, for example at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98% or 99% identity to a native polypeptide sequence. The level of sequence identity is suitably determined using the BLASTP computer program with the native polypeptide sequences as the base sequence. This means that native polypeptide sequences form the sequence against which the percentage identity is determined. The BLAST software is publicly available at http://blast.ncbi.nlm.nih.gov/Blast.cgi (accessible on 13 October 2016).
In a particular embodiment, the inhibitor is a polypeptide expressed by a nlpD gene of a bacterial species. The bacterial species is suitably a commensal bacteria or an asymptomatic carrier such as asymptomatic bacteriuria (ABU). The bacteria is an E.coli strain, such as E . coli 83972.
In a particular embodiment also, the inhibitor is a polypeptide expressed by an nldD gene and secreted by the bacteria.
In some embodiments, the inhibitor is of low molecular weight for instance less than 3kDa. It is suitably resistant to Proteinase K.
In other embodiments, the inhibitor is a larger protein, for example of about 40kDa.
Such inhibitors may be obtained by culturing the bacteria in an appropriate culture medium such as RPMI. RNA polymerase II activity, which may be tested using appropriate testing methods such as those exemplified hereinafter, may be found in the supernatant, suggesting that the factor responsible is secreted by the bacteria. Suitably the supernatant is separated from the bacteria for example using centrifugation as a preliminary step and then subjected to analysis to confirm RNA polymerase II inhibitor activity. Filtration of the supernatant for example using centrifugal or flow-through filters suitable for separating proteins can be used to remove non-active fractions which tend to be high molecular weight components, thus concentrating the inhibitor activity.
The inhibitor may be purified from the concentrate using conventional methods and the amino acid sequence determined, also using conventional methods. When this has been done, the inhibitor may be produced synthetically, for example using recombinant DNA technology.
For use in modulating the immune system, the pol II inhibitor of the invention is suitably formulated into a pharmaceutical composition in which it is combined with a pharmaceutically acceptable carrier.
The composition may be in a form suitable for oral administration, for example as a tablet or capsule, for parenteral injection (including intravesical, intravenous, subcutaneous, intramuscular, intravascular or infusion) as a sterile solution, suspension or emulsion, for topical administration as an ointment or cream or for rectal administration as a suppository.
In general the above compositions may be prepared in a conventional manner using conventional excipients.
As reported hereinafter, the mechanism of Pol II inhibition by the ABU strain was further investigated, but using re-isolates of the prototype ABU strain from inoculated patients, which were screened for attenuation of Pol II inhibitory activity. An attenuated re isolate SN25, was subjected to whole genome sequencing and genomic differences between the ancestor and progeny were identified. The variant positions in the chromosome were subsequently mutated in E . coli 83972, by homologous recombination and the mutants were screened for effects on Pol II Ser 2 phosphorylation. The genes lldD and lldR, whose products are involved in aerobic L-lactate metabolism, were shown to significantly affect Pol II phosphorylation. To confirm these effects, a biochemical approach was used. Supernatant of bacteria incubated in tissue culture medium were shown to contain inhibitory activity and was further fractionated to define the molecules responsible for these effects. Weak acids of <3 kD molecular weight, formic and acetic, were identified to have inhibitory effect of Pol II phosphorylation. In addition, Pol II phosphorylation was affected by deletion of nlpD, which encodes a lipoprotein with a potential function in cell wall formation and rfaH and cysE with products of both genes involved in biofilm formation.
In particular, the applicants have identified a new mechanism by which bacterial proteins disturb the formation of the Pol II complex and attenuate the Pol II activation in human cells. The bacterial TATA box binding protein, encoded by rpoS and its transcriptional regulator Nlpd as bacterial genes responsible for this effect through competitive inhibition of the human TATA box binding protein TBP and a reduction in PAF1C, CDK 9 and CDK12. The findings define a new, potent mechanism for cross-regulation of the transcriptional machinery between eukaryotic and prokaryotic cells. Without being bound by theory, the findings may illustrate a new general mechanism of bacterial adaptation and survival in infected hosts.
The encounters between bacteria and host cells are guided by specific molecular interactions. Pathogen attack is often executed by virulence factors via receptor-mediated interactions, involving conserved receptors like the TLRs and pathogen specific recognition mechanisms with specificity for unique sets of virulence factors. Adhesive interactions determine the tissue specificity and site of infection. Exotoxins bind cell surface receptors, such as GM1 (cholera toxin) or Gb3 (Shiga toxin) and after internalization, the toxins interfere with key cellular functions. Endotoxins activate TLR4 signaling cascades and by engaging specific co-receptors, virulence factors determine adaptor protein usage and transcription factor recruitment. Here we propose that bacteria export molecules that enter host cells and attenuate the transcriptional machinery, by competing with molecules involved in the assembly of the Pol II complex and its phosphorylation at Ser2.
As described herein, two, closely related genes have been identified in the WT strain E. coli 83972, based on a screen for "loss of function" mutants. A Tyr209Hi amino acid change in NlpD, abolished the inhibitory effect on Pol II phosphorylation. NlpD is a secreted protein with a 25 aa-signal sequence and potential signal peptidase cleavage site and is transported to the bacterial periplasm. NlpD together with other LytM (lysostaphin) - domain-containing factors is required for septal proteoglycan splitting and daughter cell separation. When overexpressed, nlpD changes bacterial morphology, due to the activation of the cell wall hydrolase AmiC. The mutation in SN25 appeared to be located within the AmiC binding site, suggesting that this mutant has lost the ability to activate AmiC and thus to facilitate the secretion of bacterial components and their interactions with the host cell. The rapid reduction in PAF1C and CDK12 protein levels suggested that host proteases or ubiquitinases might be activated. Uehara et al., 2010 discuss four possible mechanisms of bacterial amidase activation by LytM factors including NlpD. These include 1) allosteric or 2) covalent modification (e.g. proteolytic processing) of amidases by the LytM factors, 3) facilitated substrate association of the amidases and 4) prior deformation or hydrolysis of bonds in the PG substrate. Therefore, similarly to activation of the amidase in bacterial cells, NlpD might activate some other enzymes in the host cells.
The NlpD protein is of SEQ ID NO 4:
Interestingly, NlpD is homologous to a human protein, the membrane-spanning 4-domains protein, subfamily A, member 15. A putative role of the membrane-spanning 4-domains protein based on data-mining is signal transduction by being a component of a multimeric receptor complex (human gene database GeneCards). Homologous region is located towards N-terminus of NlpD (amino acids 44-90) with homology and similarity averaging 37% and 53%, respectively. This might provide some mechanistic explanation of NlpD being potentially inserted into membrane of the host cell before reaching its targets inside the cell.
NlpD also exerts its effect through transcriptional control of rpoS and rpoS-dependent genes. The transcription of rpoS is regulated from a common nlpD promoter and from additional sites within the nlpD ORF. Sigma S is an important regulator of more than 20 stationary phase genes and operons such as genes required for multiple-stress resistance. In addition, as NlpD and Sigma S proteins are encoded by the same gene cluster and are derived from the same polycystronic RNA, direct interactions cannot be excluded. It can be hypothesized that NlpD allosterically modifies Sigma S, facilitating its transport and rendering it active in binding and melting of host cell DNA.
In a particular embodiment, the inhibitor is a bacterial NplD protein, or an active variant thereof, the variant comprising an amino acid sequence having at least 70% sequence identity with the native protein. Examples of such proteins are of SEQ ID NO 4.
Without being bound by theory, it is possible that disruption of the pre-initiation complex by Sigma S might be a key step, affecting downstream transcriptional activity. The applicants have shown that bacterial RpoS competes with human TBP for binding to TATA box DNA. By dislodging TBP from its binding site, RpoS may thus prevent pre-initiation complex formation and therefore the binding of Pol II to specific promoters. A general effect on gene expression is supported by a clinical study, showing reduced expression of >60% of all genes in circulating blood cells in patients inoculated with E . coli 83972. Despite the inhibition of a large number of genes, effects were much less pronounced than when a pharmacological inhibitor was used, however, suggesting specificity. In eukaryotic promoters, PIC formation and binding of general transcription factor II B (TFIIB) recruits Pol II to the promoter. II B (TFIIB) consists of 4 functional domains - N-terminal Zn ribbon, B reader, B linker and Core domain. Core domain in eukaryotes is required to stabilize the TBP-DNA complex and the Zn ribbon recruits RNA Pol II.
Amino acid analysis reveals that Sigma S shows homology to the core domain of TFIIB (38% over the stretch of 35 AA) but no homology with the Zn ribbon, potentially explaining why Sigma S binds DNA but fails to recruit eukaryotic Pol II. The findings suggest an interesting evolutionary model, where maintaining the basic organization of gene expression and Pol II machinery constituents from bacteria to humans offers a mechanism for coordinate regulation of gene expression between eucaryotes and procaryotes.
To summarize, the applicants obtained a set of data supporting the hypothesis that both bacterial proteins NlpD and Sigma S are internalized by the human host cells. These include experiments on whole cell lysates of cells infected with ABU, co-immunoprecipitation of Pol-II, Sigma S and Nlp D, as well as immunofluorescence studies of ABU infected kidney cells (Figures 6A-6D). With immunofluorescence, some background staining is observed for NlpD and RpoS in uninfected control, which might be explained by the relative homology between NlpD and RpoS with some human proteins. NlpD is homologous to human protein, membrane-spanning 4-domains subfamily A member 15 (with 37% homology and 53% similarity over the stretch of 47 amino acids) as well to human protein GREBl-like protein isoform X4, Growth Regulation By Estrogen In Breast Cancer 1 (25% homology and 40% similarity, 116 AA). Likewise, Sigma S is homologous to two human proteins, circulating B cell antibody heavy chain variable region (31% homology and 54% similarity, 45 AA) and as discussed earlier to transcription factor II B (38% homology and 48% similarity, 35 AA).
The invention will now be particularly described by way of example with reference to the accompanying Figures in which:

Figure 1 illustrates inhibition of eukaryotic RNA Pol II phosphorylation by 83972 ABU strain: A. Cells were infected in suspension, labelled for Pol II-Ser2 with fluorescently labeled AB and run on cell flow cytometer. The distribution of phosphorylated Pol II fluorescence was analyzed. Infection with the AU strain decreases host Pol II phosphorylation; B Pol II phosphorylation in control and ABU infected cells, visualized by laser-scanning, slides were mounted and imaged. Nuclei were counterstained with DRAQ5 and fluorescence intensity was quantified by ImageJ software 1.46r.
C. Mean value of Pol II phosphorylation in ABU infected cells and uninfected control cells. One representative experiment is shown, measured with flow cytometry as shown in A and one was quantified by confocal microscopy, using ImageJ software 1.46r. Means of two experiments. D-E shows how a particular re-isolate, designated SN25, has lost the inhibitory effect on RNA Pol II phosphorylation. Results show loss of Pol II phosphorylation repression by reisolate strain SN25 as compared to ancestor strain 83972 in A-C. Error bars show standard error of the mean. G. Hit map of gene expression after infection with both strains shows 465 genes upregulated and 385 genes downregulated in both ABU and SN25, 224 genes downregulated only by ABU and 2001 genes differentially regulated by SN25, but not changed by ABU. H. Moreover, host gene expression of the innate immune response genes was also activated more efficiently by SN25 than E . coli 83972. The expression of cytokines and cytokine receptors was enhanced in SN25-infected compared to ABU-infected human kidney cells. More highly regulated cytokines include CXCL1, 2, 3 and 8, CCL5 and 10, IL1B as well as CSF2, LTB and WNT5A. The results identify SN25 as a loss of Pol II inhibition mutant, with stronger pro-inflammatory effects than the parent strain, consistent with the loss of Pol II inhibition.
Figure 2 A. Schematic location of mutations in SN25 genome compared to ABU 83972, B List of specific mutations found. C. List of genes mutated in SN25 and their corresponding functions.
Figure 3 illustrates the effect on Pol II phosphorylation of ABU deletion mutant reproducing the genomic changes in SN25. The following genes were deleted from 83972 ABU genome: lldD, lldR, nlpD, rfaH, cysE, rcsB, mdoH and lrhA. These mutants were studied with flow cytometry and fluorescence scanning microscopy for level of Pol II phosphorylation; A Cells were infected in suspension, stained for Pol II-Ser2 with fluorescently labeled AB and analyzed with flow cytometry. B. Mean value of Pol II phosphorylation in control, ABU infected sample and samples infected with single gene ABU mutants as measured by flow cytometry and C. by microscopy. It is shown that upon infection with some single gene ABU mutants, host Pol II phosphorylation becomes higher compared to ABU infection implying that these genes are involved in repression of host Pol II phosphorylation. Statistical difference in level of Pol II phosphorylation was measured with T test; D-E. Experimental design aimed to identify the Pol II inhibitor; Bacteria taken from agar plates and suspended in PBS did not show inhibitory activity; RPMI incubated with 10⁹ CFU/mL of bacteria for 4 hours was collected, centrifuged to remove bacterial cells and filtered through a 0.2 µm filter. The filtrate contained significant inhibitory activity (p<0.001), suggesting that bacterial growth in cell culture medium (RPMI) induces the secretion of inhibitor(s). Screening of supernatant of mutants revealed that all single gene mutants, apart from lldD, lldR nlpD and rfaH, secrete the substance with inhibitory activity.
Figure 4 illustrates how the ABU strain inhibits the Pol II Ser2 CTD phosphorylating machinery by targeting cyclin kinase 12 and its recruiting protein PAF1C. A. Eukaryotic phosphorylation machinery consists of two cyclin-dependent kinases CDK9 and 12, that phosphorylate CTD of Pol II biggest subunit at Ser2 residue. PAF1C complex of proteins recruits CDK12 to promoter. B. Western blot of whole cell lysates after infection with ABU and SN25 showing decrease of expression of proteins required for Pol II phosphorylation. Table inset shows % inhibition of protein as mean value of 2 experiments. C. Confocal microscopy of cells infected with ABU strain and SN25 reisolate. Cells were treated with anti-PAFlc or anti-CDK12 primary antibody and corresponding fluorescently labeled secondary antibodies. Nuclei were counterstained with Draq5. D. Fluorescence intensity of staining in C was quantified by ImageJ software 1.46r. Mean values of two experiments are shown. E. To identify genes involved in PAF1c and CDK12 inhibition, cells were infected with single gene ABU mutants and level of PAF1C and CDK12 proteins was assessed with western blot. Table below specifies % of inhibition. NlpD was identified as gene that is primarily involved in suppression. F. Confirmation with confocal microscopy of the effect of attenuated PAF1C and CDK12 suppression by d NlpD mutant.
Figure 5 . Sigma S as an effector molecule suppressing host gene expression.
A. NlpD gene is located upstream of rpoS, which encodes Sigma S; the DNA binding subunit of bacterial RNA Polymerase. NlpD regulates Sigma S expression through internal promoter. B. An rpoS deletion mutant was constructed in E . coli 83972. The loss of Sigma S protein is demonstrated by Western blot analysis of bacterial lysates of strains SN25, d nlpD and d rpoS. C. The rpoS deletion mutant was used to infect human kidney cells. Confocal microscopy shows the reduction in Pol II Ser2, Paflc and CDK12 levels was attenuated compared to the ABU strain. D. Quantification of data in C. E. Comparison of bacterial and eukaryotic type II RNA polymerase. Homologous subunits are similarly shaded. F. A schematic illustration of the hypothesis for Sigma S binding to eukaryotic promoter DNA and competitive inhibition of TBP binding. G. Testing of hypothesis in E. Human and bacterial TATA box oligo-nucleotides were incubated with synthetic peptide covering the DNA-binding domain of Sigma S. Sigma S is shown to bind prokaryotic and human TATA box oligonucleotides, creating band shifts with similar mobility. Specificity was confirmed by inhibition of binding with Sigma S-specific antibodies. H. Adding of TBP with whole cell lysate to IRF3 promoter DNA leads to TBP-DNA complex. Sigma S competes with TBP for binding to IRF3 promoter as visualized by concentration dependent decrease in intensity of shifted band.
Figure 6 . Interactions of Sigma S and NlpD with the Pol II complex
A. Confocal imaging of infected human kidney cells after staining with antibodies specific for Pol II Ser2 and Sigma S. A parallel loss of nuclear Pol II staining and accumulation of RpoS in nuclear aggregates was detected. B. Detection by Western blots of Sigma S in whole cell extracts and the nuclear fraction of cells infected with the ABU strain. Sigma S was not detected in cells infected with the SN25 reisolate or nlpD or rpoS deletion mutants. C. Binding of Sigma S and NlpD to the Pol II complex is illustrated and detected by western blot after co-immunoprecipitation of whole cell lysates with antibodies specific for total Pol II. D. Confocal imaging of human kidney cells, infected with E. coli 83972WT and stained with antibodies specific for Sigma S or Pol II, with nuclear DRAQ5 counterstaining.
Figure 7 . Functional relevance confirmed with in vivo data
A. Asymptomatic bacteriuria in C57BL/6 WT mice. Mice were inoculated with 2x10⁵ CFU/mL of ABU 83972, SN25, delta-nlpD or delta-rpoS. Mice were sacrificed after 24 hours and bladder tissues were stained with a-Pol IIp Ab. B. Loss Pol II phosphorylation is apparent after infection with ABU, this is rescued after infection with SN25, d nlpD and d rpoS mutants. This confirms in vitro data on NlpD and RpoS being involved in inhibition of Pol II phosphorylation. C. Neutrophil counts of urine of mice infected with ABU, SN25, d nlpD and d rpoS mutants. Functional relevance of Pol II de-repression in SN25 infected mutant is further suggested by higher neutrophil counts.

Example 1

Inhibition of eukaryotic RNA Pol II phosphorylation by 83972 ABU strain

The productive mRNA elongation step is generally marked by the phosphorylation of Pol II carboxy terminal domain on Serine-2 residues, consequently, Ser2 phosphorylation of Pol II is a good indicator of its activation. As a starting point, flow cytometry was developed as a technology to quantify Pol II phosphorylation ( Figure 1 ). Human kidney epithelial A498 cells were infected with ABU 83972 strain for 4 hours and after fixing and permeabilization, cells were stained with antibodies against phosphorylated Ser-2 in Pol II and goat anti-rabbit secondary antibodies labelled with Alexa-fluor 488. A marked reduction in Ser 2 phosphorylation was detected, compared to uninfected cells ( Figure 1A ).
This effect was also confirmed using confocal microscopy ( Figure 1B ). Infection with E. coli 83972 induced a change in the magnitude and distribution of phosphorylation, compared to uninfected cells. The loss of Pol II staining was visible as a loss in total fluorescence (mean value of 158, 493 AU compared to 588,307 AU, p<0.001), ( Figure 1C ). In addition, a change in distribution resulted in the emergence of two peaks, with intensities of 5,522 and 256,779 AU. Uninfected cells showed one single peak with higher fluorescence intensity at 605,108 AU. Pol II inhibition was also clearly visible using confocal microscopy ( Figure 1B ). The results confirm the inhibition of eukaryotic RNA Pol II phosphorylation by E . coli 83972.
To refine the Pol II phosphorylation data for middle-size events, Pol II phosphorylation was measured for events within gate R2. A higher number of cells fell into gate R2 for control compared to ABU infected sample (76.2 and 35.2%). ABU infection causes formation of smaller cells or broken cells (nuclei) when compared to uninfected sample as was seen from the number of small-size events. When R2-gated events are taken into consideration, lower-intensity peak of Pol II fluorescence in ABU sample becomes much less prominent. Mean value of Pol II phosphorylation in control and ABU infected sample for the representative experiment was 510,668 and 222, 972 AU, respectively.

Example 2

Identification of an ABU 83972 variant SN25 with loss of Pol II inhibitory activity and its genome sequencing

Patients were inoculated with therapeutic doses of ABU 83972. The protocol for therapeutic bladder inoculation of patients with E. coli 83972 has been described previously (Agace, J Clin Invest, 1993; Wullt, Mol Microbiol, 2000 ; Sundén, J Urol, 2010). Briefly, after antibiotic treatment to remove prior infection, patients were inoculated with E . coli 83972 through a catheter (30 ml, 10⁵ cfu/ml in saline). Blood and urine samples were obtained before and repeatedly after inoculation. Throughout the colonization period, viable bacterial counts in urine were determined, monthly urine samples were collected and analyzed for IL-6 and IL-8 as well as neutrophil infiltration. Bacteria from each urine sample were verified by PCR for presence of a kryptic plasmid unique for strain 83972 and one chromosomal marker (4.7-kb deletion in strain 83972 in the type 1 fimbrial gene cluster). For further analysis, five independent colonies per urine sample were used.
Re-isolates from inoculated patients were then screened for Pol II inhibitory activity as described in Example 1. One re-isolate, designated SN25, had lost Pol II inhibitory activity ( Figure 1D ). A498 cells were infected with ABU or SN25 E. coli strain and labelled for phosphorylated Pol II as described above. Mean values of RNA Pol II phosphorylation in SN25-infected samples compared to uninfected control cells and obtained with flow cytometry and confocal microscopy, are shown in (FigureslE and 1F, respectively). The ABU strain suppressed Pol II phosphorylation by about 73%, while SN25 by only 19%, suggesting that some genes in SN25 genome, responsible for suppression of Pol II phosphorylation, might have been lost or inactivated.
Genome of SN25 was sequenced ( Figure 2A ) to identify responsible mutations, and 36 genomic changes were found compared to E. coli 83972 wt; 25 of them were in coding region with 8 resulting in amino acid change ( Figure 2B ). Among the affected genes are those, responsible for L-lactate metabolism (lldD and lldR), regulation of motility and chemotaxis (lrhA), cell wall formation (nlpD) and biofilm formation (rfaH and cysE) ( Figure 2C ). RfaH is a transcriptional anti-terminator, required for efficient expression of long chain LPS expression and hemolysin, its loss attenuates virulence of UPEC. CysE is a serine acetyltransferase, catalyzes the conversion of L-serine to O-acetyl-L-serine. Inactivation of mdoH leads to increased expression of colanic acid capsular polysaccharide. RcsB is a positive response regulator for colanic capsule biosynthesis.

Example 3

Screening of single-gene mutants to identify genes responsible for Pol II inhibition

To identify genetic determinants of Pol II phosphorylation, genes comprising the identified variant sequences were replaced in E . coli 83972 chromosome by homologous recombination with chloramphenicol resistance cassette . Deletions were validated (Uli).. The mutants were subsequently screened for effects on Pol II ( Figure 3A ). Single deletions of ΔlldD, ΔlldR, ΔnlpD, ΔrfaH and ΔcysE reduced the inhibitory effect of E. coli 83972 wt, as shown by flow cytometry and confocal microscopy ( Figure 3B-C ). Statistical difference in level of Pol II phosphorylation was measured with t test.
The lldD gene is responsible for aerobic L-lactate metabolism, whose product catalyzes the interconversion of L-lactate and pyruvate, while lldR is a regulator of the lldPRD operon. It was concluded from these data that products of both lldD and lldR genes are responsible for suppression of RNA Polymerase II phosphorylation. The low-intensity Pol II phosphorylation peak is less prominent after infection with SN25, ABU ΔlldD and ABU ΔlldR, which is in agreement with higher mean values of Pol II phosphorylation for cells infected with SN25 and ABU mutants compared to ABU. As shown, mean Pol II phosphorylation for ungated events for control cells and cells infected with ABU, SN25, ABU ΔlldD and ABU ΔlldR is 100, 78, 79, 50 and 50%, respectively. Thus, the that products of lldD (p<0.4) and lldR genes lead to inhibition of Pol II phosphorylation, with effect of lldR being statistically significant (p<0.05).
Several other mutants, which had significant effect on de-repression of Pol II phosphorylation, are 83972ΔnlpD (p<0.01), 83972ΔrfaH (p<0.01) and 83972ΔcysE (p<0.05) ( Figure 3 B and C). Gene nlpD encodes a lipoprotein with a potential function in cell wall formation. Gene rfaH encodes for a transcriptional anti-terminator, required for efficient expression of long chain LPS, hemolysin and affects biofilm formation. Gene cysE encodes for serine acetyltransferase, which catalyzes the conversion of L-serine to O-acetyl-L-serine that is the first step of L-cysteine biosynthesis from L-serine; cysE product also affects biofilm formation in E. coli K-12.

Example 4

Secretion of bacterial inhibitors of Pol II phosphorylation

In parallel with the genetic studies, a biochemical approach was taken to identify the compound responsible for the Pol II inhibitory activity. Bacteria were incubated for 4 hours in tissue culture medium (RPMI supplemented with 1 mM pyruvate). The medium was harvested after 4 hours, centrifuged at 4,000 x g for 10 min and sterile filtered to remove remaining bacteria (0.2 µm filter), before addition to human kidney cells ( Figure 3D ). E. coli are typically 2 µm long and 0.5 µm in diameter, and filtration through 0.2 µm syringe filters removes bacterial cells. Significant Pol II inhibitory activity (p=0.023) was identified in the ABU culture supernatants compared to uninfected, substituted RPMI, suggesting that growth in cell-free culture medium induced the secretion of the inhibitor(s).
As it was shown that supernatant of ABU bacteria have similar inhibitory effect on Pol II as ABU bacteria per se, we questioned if this effect is lost in the SN25 supernatant. Phosphorylation of Pol II was significantly higher (p<0.05) after incubation with SN25 supernatant compared to ABU supernatant, suggesting that the strain has lost the ability secrete inhibitors. Mutants of SN25 were therefore were grown in RPMI and their supernatants were harvested. Supernatants of lldD, lldR, nlpD and rfaH mutants had lost Pol II inhibitory activity (p<0.02 and p<0.05, respectively) compared to ABU supernatant, ( Figure 3E ) indicating that genes lldD, lldR and nldD achieve their effect via effector molecules that are secreted by the bacteria.
The inhibitory activity of the supernatant was shown to be heat sensitive (100°C, 30 min) but Proteinase K resistant. The molecular size of the inhibitory component was found to be <3kDa, by centrifugal ultrafiltration with a 3 kDa filter. Elevated levels of acetic acid and formic acid were detected in the filtrate of the ABU supernatant, using ion exchange chromatography. A rapid increase in formic-, succinic- and acetic acids was also detected by Mass spectrometry analysis of metabolites secreted by ABU upon growth in urine. Finally, high concentrations of formic and acetic acids were shown to inhibit Pol II phosphorylation (χ² test for independence compared to medium control).

Example 5

The ABU strain inhibits the Pol II Ser2 CTD phosphorylating machinery by targeting cyclin kinase 12 and its recruiting protein PAF1C

The Pol II phosphorylation complex required to phosphorylate Ser2 is assembled in several steps ( Figure 4A ). The preinitiation complex containing the TATA box binding protein (TBP), binds to DNA upstream of the transcription start site and the activated complex then recruits transcription factor IID, and the N-teminal Zink ribbon domain of TFII b is required to recruit Pol II. following the binding of TATA box binding to DNA, TBP recruits Pol II and the DNA-binding. The DNA binding subunit of Pol II anchors the preformed complex to DNA recruits Pol II to different eukaryotic promoters and the beta subunit is phosphorylated by the C-terminal domain (CTD) phosphorylation machinery. Cyclin-dependent kinase 9 (CDK9) brings the adaptor PAF1C into close proximity with Pol II and the PAF1C subunit CDC73 recruits CDK12 to the complex. CDK9 and CDK12 then phosphorylate the Pol II CTD domain, at Ser 2 (Figure 4A).
To examine how the ABU strain suppresses phosphorylation of host RNA polymerase II CTD at Ser2 residue ( Fig. 5E ), we further analyzed potential protein targets in the Pol II phosphorylation pathway. Two cyclin dependent kinases are responsible for Pol II phosphorylation, - CDK9 and CDK12 ( Fig. 4a ). CDK9 is involved as well, bringing PAF1C in close proximity to the Pol II promoter complex. PAF1C acts as a recruitment protein for CDK12.
To address how E. coli 83972 inhibits the Pol II phosphorylation machinery, CDK12, CDC73 (PAF1C subunit) and CDK9 in host cells, after infection with ABU and SN25 was quantified by Western Blot analysis ( Fig. 4B ). CDK12 and PAF1 decreased drastically after ABU infection but remained at control levels after SN25 infection. CDK9 levels were less strongly affected (40%). This effect on PAF1C and CDK12 was confirmed by confocal microscopy ( Figure 4C-D ). SN25, in contrast, did not significantly alter PAF1C, CDK12 or CDK9 protein levels compared to uninfected cells.
To address if the inhibitory activity was a secreted bacterial product, A498 cells were treated with supernatants of cells infected with the single gene mutants of the ABU strain, and CDK12 and CDC73 levels were subsequently tested in WB. The results are shown in Figure 4E . The nlpD deletion mutant had lost the inhibitory activity against PAF1 and CDK12. This was confirmed by confocal microscopy ( Figure 4F ). NlpD gene is located in one gene cluster with rpoS, encoding one of sigma subunits of bacterial RNA polymerase ( Fig. 5A ). Remarkably, the list of genes related to CDK12 and CDC73 suppression showed a direct association with the list of genes with effect on Pol II phosphorylation. In contrast, nlpD, lldD, lldR and rfaH mutations did not affect CDK12 or CDC73 and the cysE, lrhA, mdoH and rcsB deletion mutants resembled SN25.
Overall, these results indicate that nlpD and rpoS suppress Pol II phosphorylation by targeting the CDK12 and PAF1C arm of host phosphorylating machinery.

Example 6

Investigation into NlpD and RpoS

In E . coli 83972, NlpD is located upstream of rpoS, which encodes Sigma S; the DNA binding subunit of bacterial RNA Polymerase ( Figure 5A ). NlpD regulates Sigma S expression and may facilitate its release by activating cell wall hydrolases. To address if the effects of NlpD on Pol II and PAF1C are executed through rpoS, we constructed an rpoS deletion mutant in E . coli 83972. The deletion was confirmed by DNA sequencing and the loss of Sigma S protein was verified by Western blot analysis of bacterial cell extracts ( Figure 5B ). Sigma S was present in extracts from E. coli 83972WT bacteria but was absent in extracts from SN25, ΔnlpD and ΔrpoS. In bacterial supernatants, the loss of Sigma S in these strains was confirmed (Figure 5B).
The applicants subsequently examined the effect of the rpoS mutant on Pol II phosphorylation ( Figure 5C ). The rpoS mutant had lost the ability to inhibit Pol II phosphorylation, and Pol II staining in infected cells was comparable to SN25 and the delta NlpD mutant (n.s.). A similar effect was observed for PAF1C, CDK12 as the rpoS mutant had lost the ability to inhibit PAF1C and CDK12 ( Figures 6C-D ). The results suggest that Sigma S acts as bacterial effector molecules in human cells, responsible for the inhibition of Pol II phosphorylation through PAF1C. The results suggest that the effects of NlpD on RpoS account for the attenuation of Pol II -Ser 2 phosphorylation.

Example 7

Subcellular distribution of nlpD and rpoS in infected human cells

To address if these molecular interactions may be relevant in infected cells, the applicants subsequently examined, if Sigma S is internalized into human cells, after infection with E. coli 83972WT. By Western blot analysis of whole cell extracts, a single band of 40 kDa was detected, after staining with Sigma S specific antibodies. A band with similar mobility was detected in nuclear extracts, suggesting nuclear translocation of Sigma S ( Figure 6B ). The Sigma S band was not detected in cells infected with SN25 or the ΔnlpD- or ΔrpoS deletion mutants.
The internalization and nuclear translocation of Sigma S was confirmed, by co-immunoprecipitation, using Pol II specific antibodies. A band corresponding to Sigma S was detected in extracts from cells infected with ABU but not SN25. In a further Pol II co-ip involving whole cell extracts from cells infected with the NlpD or RpoS mutants, Sigma S and NLPD bands were detected in ABU infected cells but not in the single gene mutants. Low Sigma S in SN25, which does not carry a deletion.
In addition, human kidney cells were infected with E. coli 83972WT and stained with antibodies specific for Sigma S or Pol II, with nuclear DRAQ5 counterstaining. A parallel loss of nuclear Pol II staining and accumulation of RpoS in nuclear aggregates was detected. In contrast, Sigma S was not detected in cells infected with SN25, ΔrpoS or AnlpD ( Figure 6D ).

Example 8

Competitive inhibition of TBP binding by Sigma S

The Pol II phosphorylation complex required to phosphorylate Ser2 is assembled in several steps (Figure 4A). The preinitiation complex containing the TATA box binding protein (TBP), binds to DNA upstream of the transcription start site and the activated complex then recruits transcription factor IID, and the N-teminal Zink ribbon domain of TFII b is required to recruit Pol II. following the binding of TATA box binding to DNA, TBP recruits Pol II.
Like the TATA-box binding protein in eukaryotes, Sigma S binds to DNA and is the TATA box binding protein of the bacterial RNA Pol II complex ( Figure 5E ). We therefore tested the hypothesis that Sigma S may bind to eukaryotic promoter DNA and competitively inhibit TBP binding ( Figure 5F ). Human and bacterial TATA box oligonucleotides were incubated with a synthetic peptide comprising the Sigma S DNA-binding domain (aa 149-183- SEQ ID NO 2) in an electro-mobility shift assay (EMSA). Sigma S was shown to bind prokaryotic and human TATA box oligonucleotides, creating band shifts with similar mobility ( Figure 5G ). Specificity was confirmed by inhibition of the band shift with Sigma S-specific antibodies. The Sigma S peptide was subsequently shown to competitively inhibit TBP binding to the IRF3 promoter, in a concentration-dependent manner ( Figure 5H ).
To examine if E. coli 83972 affects PIC formation in infected cells, we quantified the TATA box binding protein (TBP) in total cell extracts from uninfected and infected kidney cells ( Figure 5B-D ). E . coli 83972 reduced the TBP and TF2b protein levels. By confocal microscopy, nuclear transcription factor II b (TF2b) staining was reduced. SN25, in contrast, did not affect the PIC components ( Figure 5C and D ) and the nlpD deletion mutant reproduced the SN25 phenotype ( Figure 5F ).

Example 9

In vivo relevance

The effects on Pol II phosphorylation were confirmed in vivo, in the murine urinary tract infection model. C57BL/6 WT mice were inoculated with 2x10⁵ CFU/mL of ABU 83972, SN25, delta-nlpD or delta-rpoS and sacrificed after 24 hours. Tissue levels of RNA Pol II were quantified by staining of frozen tissue sections after staining with specific antibodies ( Figure 7A ). E. coli 83972 inhibited mucosal Pol II staining in the urinary bladder mucosa, confirming the cellular studies (p<0.05, Figure 7B ). This inhibitory effect was not detected in mice infected with the SN25 mutant strain or the delta NlpD or Sigma S mutant strains. Neutrophil counts in urine of infected mice were measured (Figure 7C) and higher counts were found in SN25 infected mice, indicating functional relevance of Pol II de-repression. Furthermore, it was found that the SN25 infection led to higher level of bacteria in bladder, suggesting higher virulence.

MATERIALS AND METHODS

Bacterial strains

Asymptomatic bacteriuria (ABU) strain was isolated during a study of childhood UTI in Göteborg, Sweden (Lindberg et al., 1978). Bacteria were cultured on tryptic soy agar (TSA, 16 h, 37°C) and harvested in phosphate-buffered saline (PBS, pH 7.2). For the course of infection, bacteria were diluted to reach final concentration in medium 1x10⁸ cfu/ml.

Cell culture

Human kidney carcinoma (A498, ATCC HTB44) were cultured in RPMI-1640 (Thermo Scientific) supplemented with 1 mM sodium pyruvate, 1 mM non-essential amino acids (GE Healthcare) and 10% heat-inactivated FBS at 37°C with 5% CO₂.

Preparation of bacterial supernatant

Bacteria were incubated for 4 hours in tissue culture medium, the medium was harvested, centrifuged at 4,000 x g for 10 min and filtered to remove remaining bacteria. Human kidney cells A498 were incubated with filtered supernatant for 4 hours.

Cell flow cytometry

Before infection, A498 cells were washed twice with RPMI medium without FCS. Cells were detached with Versene for 15 min, centrifuged at 400g for 5 min and re-suspended in ice-cold PBS. 5x10⁵ cells were treated in suspension as follows: cells were infected, fixed (3.7% formaldehyde in PBS, 15 min), permeabilized (0.25% Triton X-100, 5% FBS in PBS, 10 min), blocked (5% FBS, 1h at RT), incubated with primary antibodies in 5% FBS overnight at 4°C (anti-RNA Polymerase II subunit B1 (phospho CTD Ser-2) 1:800, Merck) and with fluorescently labeled secondary antibody (Alexa Fluor^® 488 goat anti-rat IgG, A-11006, 1:600, Thermo Scientific) for 1h at RT. After each step above apart from permeabilization and blocking, cells were washed twice with ice-cold PBS and centrifuged at 400g for 5 min. After final wash, cells were re-suspended in flow cytometry buffer 0.02 % EDTA 5% FCS in PBS. With BD Accuri C6 flow cytometer (BD Biosciences), 20,000 events were collected at 60 ul/min flow rate.

Confocal microscopy

Cells were grown to 70-80% confluence on 8-well chamber Permanox^® slides (3x10⁴ cells/well, Thermo Fisher Scientific), and infected, fixed, permeabilized and treated with AB as for flow cytometry. After nuclear staining (15 min, DRAQ5, Abcam), slides were mounted (Fluoromount, Sigma-Aldrich), imaged by laser-scanning microscopy (LSM800, Carl Zeiss) and quantified by ImageJ software 1.46r (NIH).

Ion exchange chromatography

Organic acids were analyzed on a Dionex anion chromatography system by the Swedish Environmental Research Institute. Potassium hydroxide was used as an eluent to separate ions in the sample. To obtain the best possible separation the concentration of the eluent was gradually changed during the process. After separation, a cation exchanger was used to reduce the conductivity of the eluent and to convert the anions into their respective acids.

Global gene expression

Total RNA was extracted from A498 cells in RLT buffer with 1% β-Mercaptoethanol. 100 ng of RNA was amplified using GeneChip 3'IVT Express Kit, 6 ng of fragmented and labeled aRNA was hybridized onto Human Genome array strips for 16 hours at 45°C, washed, stained and scanned using the GeneAtlas system (Affymetrix). All samples passed the internal quality controls included in the array strips (signal intensity by signal to noise ratio; hybridization and labeling controls; sample quality by GAPDH signal and 3'-5' ratio < 3).
Fold change was calculated by comparing cells treated with ABU or mutants to uninfected cells (PBS control) of the same genetic background. Significantly altered genes were sorted by relative expression (2-way ANOVA model using Method of Moments, P-values < 0.05 and absolute fold change > 1.41) (Eisenhart 1947). Heat-maps were constructed with Excel. Differentially expressed genes and regulated pathways were analyzed by Ingenuity Pathway Analysis software (IPA, Ingenuity Systems, Qiagen) and String and David open source software.

Western blotting

Cells were lysed with RIPA lysis buffer, supplemented with protease and phosphatase inhibitors (both from Roche Diagnostics). Proteins were run on SDS-PAGE (3-8 % or 4-12% Bis-Tris gels, Invitrogen), blotted onto PVDF membranes (GE Healthcare) blocked with 5% non-fat dry milk (NFDM), incubated with primary antibody: mouse anti-CDK12 (1:400 in 5% NFDM, ab9722, Abeam), mouse anti-Parafibromin (1:400 in 5% BSA, sc-22514-R, Santa Cruz), washed with PBS tween 0.1 % and incubated with secondary antibodies in 5% NFDM (goat anti-mouse-HRP, Cell Signaling). Bands were imaged using ECL Plus detection reagent (GE Health Care) and quantified using ImageJ. GAPDH (1:1,000, sc-25778, Santa Cruz) was used as loading control.

SEQUENCE LISTING

<110> Linnane Pharm AB
<120> Novel Therapy
<130> P3095/WO
<150> GB1617548.1
<151> 2016-10-17
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<223> Sigma S protein
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<212> PRT
<213> Escherichia coli
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<223> Sigma S protein
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<211> 35
<212> PRT
<213> Escherichia coli
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<210> 4
<211> 379
<212> PRT
<213> Escherichia coli
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<223> NplD protein
<400> 4

Claims

An inhibitor of RNA polymerase II for use in therapy, wherein said inhibitor is a moiety which targets a protein selected from cyclin kinase 12 (CDK12) or its recruiting protein PAF1C, and wherein said inhibitor is (a) an E. coli NlpD protein, or an active variant thereof, the variant comprising an amino acid sequence having at least 70% sequence identity with the native protein, or (b) an E. coli Sigma S protein or an active variant thereof, the variant comprising an amino acid sequence having at least 70% sequence identity with the native protein, or an active fragment of either of these, the active fragment comprising SEQ ID NO 3.
An inhibitor of RNA polymerase II for use according to claim 1 wherein the E. coli is a commensal bacteria or an asymptomatic carrier, preferably wherein the E. coli is asymptomatic bacteriuria (ABU), yet more preferably wherein the E. coli are E. coli 83972.
An inhibitor of RNA polymerase II for use according to claim 1 which is a Sigma S protein or a variant thereof or an active fragment of either of these, preferably wherein the Sigma S protein is of SEQ ID NO 1 or SEQ ID NO 2 or a variant thereof, or an active fragment of either of these, the active fragment comprising SEQ ID NO 3.
An inhibitor of RNA polymerase II for use according to claim 1 which is a bacterial NlpD protein, or a variant thereof, preferably which is a protein of SEQ ID NO 4.
An inhibitor of RNA polymerase II for use according to claim 2 wherein the inhibitor is secreted by the E. coli.
An inhibitor of RNA polymerase II for use according to any preceding claim, wherein the NlpD protein is of less than 3kDa in molecular weight.
An inhibitor of RNA polymerase II for use according to any one of claims 2 to 6 which is synthetic.
An inhibitor of RNA polymerase II for use according to any one of the preceding claims wherein the therapy is immunosuppression, anti-inflammatory or anti-infection therapy.